Since polyclonal and monoclonal antibodies have the ability to specifically bind to trace proteins contained in a mixed solution such as blood, they are industrially useful materials as reagents in researches and/or clinical tests or as pharmaceutical preparations, etc.
Antibody molecules strongly induce antigen-antibody reaction against antigens expressed on the cell membrane, and hence desired antibodies are those recognizing membrane proteins in order to expect a therapeutic effect when administered to diseased patients.
Antibodies which have been marketed for use in disease treatment are mostly monoclonal antibodies recognizing membrane proteins (Non-patent Document 1), and they produce a therapeutic effect through their actions such as (1) binding to a target cell to induce cell death directly or indirectly (Patent Document 1) or (2) inhibition of ligand binding to membrane receptors to reduce intracellular signaling (Patent Document 2), etc.
Moreover, antibodies which have been marketed are each obtained as a result of repeating inventive efforts and enormous works.
Various attempts have been made to develop techniques for obtaining antibodies recognizing membrane proteins. However, no simple method has been established for preparation of monoclonal antibodies against membrane proteins.
For example, there is a known method in which a peptide sequence exposed on the cell membrane surface is prepared by forced expression in E. coli or other cells or by chemical synthesis, and the peptide sequence thus prepared is attached to a carrier protein to thereby induce immune responses.
However, such a method fails to cause three-dimensional structure formation and/or post-translational modifications (e.g., with sugar chains), and hence it is difficult to yield antibodies recognizing the inherent structure of membrane proteins.
To obtain monoclonal antibodies against membrane proteins, the membrane proteins should retain their inherent three-dimensional structure during immunization into laboratory animals. However, when a surfactant is used for extraction of membrane proteins from the cell membrane, the membrane proteins lose their three-dimensional structure. Or alternatively, when no surfactant is used for this purpose, the membrane proteins aggregate through their hydrophobic regions. Because of these problems, it is not easy to prepare membrane proteins retaining their three-dimensional structure for use as antigens.
As an immunization method using a full-length protein, a genetic immunization method is reported, in which a DNA vector for expression is directly introduced into mice (Patent Document 3). This method has many advantages, particularly in that it requires no purified antigen and in that it yields antibodies recognizing the three-dimensional structure of a target molecule.
However, such a method is known to have problems to be solved, for example, in that (1) the transgene should be expressed on the cell surface in order to induce immune responses, (2) sufficient immunization cannot be achieved due to low expression level of the transgene, (3) if the extracellular region is too small, the immune system cannot respond and hence antibodies are difficult to produce, and (4) it is difficult to obtain antibodies against multi-transmembrane proteins among membrane proteins (Non-patent Document 2).
In another method reported, a functional membrane protein is reconstructed on the baculovirus membrane and used for immunization (Non-patent Document 3).
However, the above document shows that this method causes weak immune responses during induction of antibody specific to a target molecule. Moreover, such a method results in post-translational modifications (e.g., addition of complex N-linked sugar chains) different from those found in mammalian cells because baculovirus is prepared in insect cells, and there is a lot of uncertainty as to whether all membrane proteins can take their functional structure on the viral membrane.
To eliminate the need for considering the problems of three-dimensional structures, some methods are reported, which involve direct administration of grown cells to mouse tail vein or abdominal cavity to cause immune responses (Patent Documents 4 to 6 and Non-patent Documents 4 to 8). In these methods, 1 to 10×106 cells (corresponding to about 4 to 40 mg protein), which are larger than the normal antigen dose (100 to 200 μg target protein), are administered for a short period of time (at intervals of 1 week) and antibody-producing cells are collected from 1 to 6 weeks after the initiation of immunization.
For cell administration, an improved method is reported in which human cancer tissue is administered under the skin or into the gonadal fat pad in mice (Patent Document 7).
However, the methods disclosed in Patent Documents 4 to 6 and Non-patent Documents 4 to 8 require further studies for their practical use in the following points: (1) immune responses are difficult to occur against a specific substance because many kinds of proteins are immunized at the same time; (2) anaphylactic shock will be caused because proteins are administered in large amounts; and (3) animals are more likely to die earlier, e.g., due to organ failure caused by metastasis of cancer cells.
Moreover, to obtain many types of high-affinity antibodies, it is important to ensure the progress of hyperimmunization. In conventional methods, it is known that immunization with a lower antigen dose for a longer period of time is more likely to facilitate hyperimmunization. For example, multiple administrations (empirically 5 or more) at intervals of 3 weeks or longer are known to be preferred for mice (Non-patent Document 9). Hyperimmunization refers to a combination of the following three mechanisms: class shift to IgG; affinity maturation, and clonal dominance (apoptosis-induced selective death of antibody-producing cells with low specificity), and it is known as a mechanism which allows selective proliferation of high-affinity antibodies (Non-patent Document 9).
In the methods disclosed in Patent Documents 4 to 6 and Non-patent Documents 4 to 8, mice are more likely to die earlier and hyperimmunization is less likely to proceed, because a large amount of cells are administered for induction of immune responses. It is therefore difficult to sufficiently induce effective immune responses by the methods for direct cell administration disclosed in these documents.
Further, in the method disclosed in Patent Document 7, (1) it is necessary to isolate human tissues containing B cells, (2) immune responses are not reproducible due to differences in the types of cells contained in the tissues, and (3) in vitro culture is difficult and gene transfer or other techniques are also difficult to apply.